Process for the 1,2-dehydrogenation of a steroid with septomyxa



United States Patent PROCESS FOR THE 1,2-DEHYDROGENATION OF A STEROID WITH SEPTOMYXA Adolph Weintraub, Brooklyn, N.Y., and Samuel H. Eppstein, Kalamazoo, and Peter D. Meister, Kalamazoo Township, Kalamazoo County, Mich., assignors to The Upjohn Company, Kalamazoo, Mich., a corporation of Michigan No Drawing. Application September 26, 1957 Serial No. 686,295

26 Claims. (Cl. 195-51) The present invention relates to a novel process for the fermentative dehydrogenation of steroids, the oxidative scission of the C-17 side chain of steroids, or both, and the products resulting therefrom.

This application is a continuation-in-part of application S.N. 493,302, filed March 9, 1955, now abandoned.

The process of the present invention comprises subjecting a steroid to the action of a fungus of the genus Septomyxa. Dehydrogenation is thereby effected especially at the 1,2-position when the starting steroid is saturated at the 1- and Z-position carbon atoms. Of special interest for dehydrogenation are starting 3-oxygenated steroids having two hydrogen atoms at each of the 1- and 2-position carbon atoms. Degradation of the 17-side chain may also be effected to give 17-hydroxy and l7-keto steroids, especially from a starting 20-oxygenated steroid.

Whereas side chain degradation and concomitant ring cleavage have been observed with a variety of organisms, dehydrogenation in ring A and selective side chain degradation without ring cleavage has been observed with but a small number of organisms. Thus Fried et al,, I. Am. Chem. Soc. 75, 5764 (1953), shows that Cylindrocarbon radicola fermentation of progesterone, 17a,21-dl hydroxy-4-pregnene-3,ZO-dione, or testosterone produces l-dehydrotestololactone with cleavage of the original D ring. In contradistinction thereto, the instant Septomyxa fermentation of steroids permits the D ring in a large measure to remain intact.

It is an object of the present invention to provide a Septomyxa fermentation method for the dehydrogenation of steroids, especially steroids saturated at the 1,2-position. Another object of the invention is the provision of a process for the production of l-dehydro steroids. Another object of the invention is to provide a method for the degradation of the 17-side chain of steroids, especially 20-oxygenated steroids, by the action of a fungus of the genus Septomyxa. A further object of the invention is the provision of a process for the production of 17-hydroxy-steroids and 17-ket0steroids. Additional objects of this invention are the provision of new and useful l-dehydro steroids. Other objects and uses of this invention will be apparent to those skilled in the art to which this invention pertains.

The production of l-dehydro steroids is known, but heretofore it has entailed diflicult chemical processing such as dibrornination of a steroid to give the 2,4-dibromo compound, along with other isomers, which could then be dehydrohalogenated with pyridine to give a 1,4-dehydro steroid, Fieser and Fieser, Natural Products Related to Phenanthrene, ed. .3, page 264, Reinhold Publ. Co., N.Y., 1949, and Djerassi US. Patent 2,579,479 of December 25, 1951. By the present process, the l-dehydro and 1,4-dehydro steroids can be made directly.

The instant process is useful to reintroduce the 1-dehydro function where, for example, it may have been destroyed by previous processing such as by yeast l-dehydrogenase incubations, which converts l-dehydroandrostene-3,17'dione to androstane-3p,17p-diol with saturation of the 1,2-position. Butenandt, Ber. 73:818 (1940). Heretofore, direct changes within this recognized group of l-dehydro steroids have not been available because of lack of a suitable method of preparing l-dehydro steroids, Dorfman and Ungar, Metabolism of Steroid Hormones, pages 86 and 93, Burgess Publ. Co., Minneapolis, Minn., 1953.

The method of the present invention is able to furnish a variety of products which are useful therapeutics per se as well as being useful as intermediates in the production of therapeutic products. The instantly produced l-dehydro steroids as compared with the corresponding 1- saturated steroids commonly have a lower melting point and enhanced pharmacological activity. They are additionally valuable intermediates where it is desired to aromatize the A ring.

The starting compounds of the present invention are either amenable to dehydrogenation, especially at the 1,2- position, or degradation of the 17-side chain, or both. Dehydrogenation is of special interest in conjunction with steroids saturated at the 1,2-position. Dehydrogenation can be effected preferably upon such steroids which are 3-oxygenated, that is containing the 3-hydroxy, B-ether, 3-ester, or 3-keto group, in which the 1,2,3-position carbon atoms may be represented as follows:

CH CH CO--; -CH -CH -CHOH--; and CH -CH(CH )-CO- Of special interest in conjunction with degradation of the 17-position side chain are 20-oxygenated starting steroids, preferably the ZO-hydroxysteroids and 20-ketosteroids. The starting cyclopentanopolyhydrophenanthrene can possess other substituents, for example, keto groups or hydroxy groups in positions 3, 6, 7, 8, 11, 12, 14, 17 and 21, particularly in the ll-position, and can have double bonds in various positions especially in positions 4,5 and 7,20. The starting steroid can have various substituents elsewhere on the molecule without impairing the nature of the reaction upon the herein described active positions. Where the starting steroid is saturated in the 1,2-position, the steroid may be either degraded, in the 17-position side chain, to 17-hydroxy or 17-keto, or dehydrogenated at the 1,2-position to give a double bond at the 1,2-position, or both degraded at the side chain and dehydrogenated. Aromatic A ring steroids result from a starting 19-hydroxy, 19-aldehyde or 19-normethyl-3-keto-4-dehydro steroids. Representative starting materials include: 304- and 3fi-hydroxypregnane-ZO-one, 3aand 3B-hydroxy-5-pregnene-20-one, 3aand 3,B-hydroxy pregnane-11,20-dione, 30,llcc, 3al1/3-, 35,111 and 3 8,1 1 ,8-dihydroxypregnane-20-one, ll-dehydroprogesterone, 9a-fiuoro-, 9a-Chloroor 9ot-bromo-progesterone, 17ix-methyldehydrocorticosterone, 17u-methyl-l1-desoxycorticosterone, 17cc methylprogesterone, 12a hydroxy progesterone, progesterone, ll-ketoprogesterone, Ila-hydroxyprogesterone, lla-acetoxyprogesterone, 11,8-hydroxyprogesterone, 14a-hydroxyprogesterone, 17a-hydroxyprogesterone, l4oz,l7zx,2l trihydroxy-4-pregnene-3, 20 dione, 14a,17a-dihydroxy-21-acetoxy-4-pregnene-3,20- dione, 11a,17a-dihydroxyprogesterone, 17a-hydroxy-llketoprogesterone, 3 8,170:,20-allopregnanetriol, c,2l-dihydroxy-4-pregnene, 3,20-dione, 21-dihydroxy-4-pregnene- 3,11,20-trione, corticosterone 11,9,21-dihydroxy-4-pregnene-3,20-dione), ll-desoxycorticosterone (21-hydroxy- 4-pregnene-3,20-dione), cortisone (17a,21-dihydroxy-4- pregnene-3,11,20-trione), cortisone acetate (17ot-hydroxy- 2 1-acetoxy-4-pregnene-3 ,1 1,20-trione) hydroxycortisone (11,8,17a,21-trihyclroxy-4-pregnene-3,20-dione), 9a-Chl0- ro-, bromoor fiuoro-l1/3,17a,21-trihydroxy-4-pregnene- 3,20-dione, 2-methyl-11fi,17a,21 trihydroXy-4-pregnene- 3,20-dione, 1 1a,17a,21-trihydroxy-4-pregnene-3,20-dione, 4-androstene-3,17-di one adrenosterone (4 androstene- 3,11,17-trione), ll-ketotestosterone, 11v:- and 11 ,a-hydroxy 17cc methyltestosterone, 17u-ethinyltestosterone, pregnane-3,l1,20-trione, 17cc hydroxypregnane-3,1 1,20 trione, 17a,2l-dihydroxypregnane 3,11,20-trione, pregnane-3,l2,20,-trione, pregnane 3,20-dione, l7oc-hydroxypregnane 3,2'0-dione, aIlopregnane-IH1,20-trione, allopregn'ane 3,20 dione, 30c hydroxy S-pregnene-ZO-one, 313-1la-dihydroxyallopregnane-20-one, Zia-and 3e-hydroxye allopregnane-20-one, androstrone (androstan-3arol+l7- 6. 1 3'a,l212,21 trihydroxypregnane-ZO-one, testosterone, 19 V normethyltestosterone, 17a methyltestosterone, llflZl-dihydroxyl, 17 20) -pregnadiene-3-one, 2-methyl- 113,21 dihydroxy 4,17(20) pregnadiene-3-one, 21-hyirony-4, l7(2 0),pregnadiene -3, 1 l-dione, 1 la,2 l-dihydroxy.- 4,17 (20) -pregnadiene-3-one, 2-bromo-l l,B,2l-dihydroxyr 4,l7(20) r pregnadiene-3-one, l7ot-2l-dihydroxy-4,9(ll)- pregnadiene 3 ,2 dione, 17ot,21-dihydroxyr4-prcgnene- 9,:Il-r3 L oxido 3,20 dione, 2-methyl-l7a,2 l-dihydroxy- 4,9(11)-pregnadiene-3,20-dione, 2 methyl 17a,21-dihy d roxy-4-pregnene-9:11-/3-oXido-3,20-dione, and Z-methyl- 9a-chloro-, bromo-, or fluoro-l1B,17a,2l-trihydroxy-4- pregnene3,20-dione. Either the free hydroxy steroids or their ester form can be used as starting steroid substrate. The resulting 1,2-dehydrogenation takes place in any event to produce the corresponding l-dehydro analogue of the starting material, recovered as the free alcohol.

In the process of the present invention, the operational conditions and reaction procedure and details may be those already known in the art of steroid bioconve rsion as illustrated by the Murray et al. US. Patent 2,602,769, issued July 8, 1952, utilizing however the action of a species of fungus of the genera Septomyxa. The genus Septomyxa belongs to the class of Deuteromyces, Fungi Imperfecti, of the order Melanconiales, of the family Melanconiaceae. Among the species of the genus Septomyxa which are. useful in the fermentation of steroids are Septomyxa afiinis, Septomyxa aesculi, Septomyxa corni, Septomyxa salicina, and Septomyxa tulasnei. Typical strains suitable for the practice of the invention are the following: i i Septomyxa afiinis (Sherb.) Wr., ATCC 6737. Septomyxa ajfinis, Centraalbureau voor Schimmelcultures at Baarn, Holland (strain from v. Eck and from Wollenweber). Septomyxa corni Oud., Centraalbureau voor Schimmelcultures at Baarn, Holland (strain from v. Arx). Septomyxa aflinis (Sherb.) Wr., Quartermaster 40b.

The foregoing cultures are listed in current catalogs (e.g. ATCC 6737, ATCC 13414, ATCC 13425, ATCC 13416 and Quartermaster 40b) and are suitable for the practice of the invention. It is to be understood, however, that other Septomyxa strains of these species or the typical species mentioned above are suitable.

Culture of the fungi, for the purpose and practice of the present invention, is in or on a medium favorable to the development of the fungi. Solid media can be utilized, but the preferred media are those which permit quantity growth under aerobic conditions. Moist solid particulate media, such as bran, cereal grains, cereal grits, wood chips, shavings, sawdust, cornhusks, fibrous material, such as copra, chestnuts, or lupine seeds may be used These can be extracted with alcohol, ether or other organic solvents, to remove objectionable contaminants and growth inhibitors prior to fermentation, The carriers can optionally contain added growth factors and nutrients and can be used in layers or trays with or without auxiliary aeration, in towers or in the vinegar process or under conditions of agitation as for example by tumbling in a rotating drum. Liquid media, illustratively brewers wort, are well adapted to use under aerobic layer or more especially aerobic submerged fermentation conditions. Suitably the media should contain sources of available carbon, nitrogen and minerals although of course there can be significant growth and deve p n un e le s th optimum conditions.

Available carbon can be from carbohydrates, starches, gelatinized starches, dextrin, sugars, molasses as of cane, beet and sorghum, glucose, fructose, mannose, gelactose, maltose, sucrose, lactose, pentoses, amino acids, peptones or proteins, the hexoses being preferred. Lower fatty acids, higher fatty acids, or fats are illustrative of other materials which provide assimilable carbon for the energy requirements of the fungi. Mixtures of various carbon sources are sometimes advantageous.

Nitrogen in assimilable form can be provided by soluhis or insoluble vegetable or animal proteins, soybean meal, lac bu n, cas gg lb mi pep nes, p ypeptides, amino acids, acid amides, urea, ammonium salts, ammonia trapped on base exchange resins or on zeolites, ammonium chloride, nitrates, sodium nitrate, potassium nitrate, or morpholine. Whey, distillers solubles, corn steep liquor, or yeast extract have been useful.

As mineral constituents the media or menstruum can contain, naturally present or added, available aluminum, calcium, chromium, cobalt, copper, gallium, iron, magnesium, molybdenum, potassium, scandium, uranium, and vanadium. Sulfur can be provided by sulfates, alkyl sulfonates, sulfoxylates, sulfamates, sulfinates, free sulfur, hyposulfite, persulfate, thiosulfate, methionine, cystine, cystein, thiamin, or biotin. Phosphorous, preferably pentavalent, suitably in a concentration at or about 0.001 to 0.07 molar and particularly at or about 0.015 to 0.02, can be present, suitably as ortho-, meta-, or pyrophosphates, salts or esters, phytin, phytic acid, phytates, glycerophQ Phates, sodium nucleinate, and/ or corn steep liquor, casein, lecithin or ovovitellin. Boron, iodine and selenium in traces may be advantageous. Desirably boron, in the form of boric acid or sodium borate can be presout or added especially after germination and early growth of the fungus.

Other accessory growth factors, vitamins, auxins and growth stimulants can be provided as needed or desired.

While solid or liquid media can be utilized, a liquid medium is. preferred as it favors mycelial growth.

To guard against infection the fermentation medium can contain added antiseptic or antibiotic agents such as benzoates, sulfites, penicillin, or tetracycline.

' Suspending or mycelial carriers such as filter earths, filter aids, finely divided cellulose, wood chips, bentonite, calcium carbonate, magnesium carbonate, or other suspendable solid matter, substances such as methyl cellulose, carboxyrnethyl cellulose, alginates or polyvinyl alcohol can be added to facilitate fermentation, aeration and, filtration.

The selected species of fungus is grown on a medium suitably containing assimilable non-steroidal carbon, illustratively carbohydrates, such as dextrose; assimilable nitrogen, illustratively soluble or insoluble proteins, peptones or amino acids, and mineral constituents, illustratively sodium or ammonium phosphate and magnesium sulfate. The medium can desirably have a pH before inoculation of. between about four to about seven though a higher or lower pH may be used. A pH of between about five and about six ispreferred for the growth of Septomyxa. Inoculation of the fungi growth-supporting medium with the selected species of Septomyxa may be accomplished in. any suitable manner. SeptomyXa grow over a range from about twenty to about 38 degrees centigrade with a temperature between about twenty to 3-5 degrees centigrade preferred.

The development period of fungal growth required before the steroidto be fermented. is exposed to the fungus does not appear to be critical, for example, the steroid can be added either before thermal or other sterilization ofthe medium, at the time ofinoculating the medium with a selected SeptomyXa species, or at sometime, for example, 24 or48 hours later. The steroid to be fermented can be added at any suitable concentration although for practical reasons steroid substrate at a concentration of about or up to about 0.5 gram per liter or even 0.8 gram per liter of medium is satisfactory and two grams per liter is operative although higher concentration, depending on the particular steroid, can be used. The addition of steroid substrate to be fermented can be accomplished in any suitable manner especially so as to promote a large surface of contact of the steroid substrate with the fungus, such as by dispersing the steroid substrate, either alone with a dispersing agent, or in solution in an organic solvent by mixing or homogenizing a steroid substrate with a fungal medium to form a suspension or dispersion of the steroid. Either submerged or surface culture procedures can be used with facility, although submerged culture is preferred. Alternatively, steroid fermenting enzymes of a growth of the fungus can be separated from the fungus or medium, admixed with the steroid or a solution or dispersion thereof, and the mixture subjected to aerobic conditions to accomplish fermentation of the steroid.

The temperature during the period of fermentation of the steroid can be the same as that found suitable for fungal growth. It need be maintained only within such range as supports life, active growth, or the enzyme activity of the fungus.

While any form of aerobic incubation is satisfactory for the growth of the selected fungus and fermentation of the steroid substrate, the efficiency of steroid fermentation is related to aeration and agitation. Therefore, aeration is usually controlled, as by agitation and/or blowing air through the fermentation medium. Aeration can be effected by surface culture or under submerged fermentation conditions. Aerobic conditions include not only the use of air to introduce oxygen, but also other sources or mixtures containing oxygen in free or liberatable form. In using air as the aerating medium, a desirable rate of aeration is about four to twenty millimoles and particularly about nine to twelve millimoles of oxygen per hour per liter as determined by the method of Cooper, Fernstrom and Miller, Ind. Eng. Chem. 36, 504 (1944). Aeration is suitably modified by using superatmospheric or subatmospheric pressures, for example, thirty pounds per square inch or ten pounds per square inch absolute. Oxygen uptake can be facilitated by the presence of various agents such as ascorbic acid, glutamic acid, citric acid, lactic acid, tyrosine, or tryptophane. The pH may be controlled by addition of alkali or phosphoric acid. The addition of excess calcium carbonate to maintain a solid calcium carbonate residue has been found desirable.

The time required for the fermentation of steroid varies somewhat with the procedure. When the steroid substrate is present at the time of inoculation of the medium, periods of from eight to 72 hours may be used. However, when the steroid is added to the fungus, after substantial aerobic growth of the fungal organism, for example, after sixteen to 24 hours at optimum temperature, the conversion of steroid substrate begins immediately and high yields are obtained in from one to 72 hours, 24 hours being generally satisfactory.

After completion of the steroid fermentation, the resulting fermented steroid is recovered from the fermentation reaction mixture. An especially advantageous manner of recovering the fermented steroid involves extracting the fermentation reaction mixture, including the fermentation liquor and mycelia with a water-immiscible organic solvent for steroids, for example, methylene chloride, chloroform, carbon tetrachloride, ethylene chloride, trichloroethylene, ether, amyl acetate, benzene, and the like. The fermentation liquor and mycelium can be separated and then separately extracted with suitable solvents. Mycelium can be extracted with either watermiscible or water immiscible solvents, acetone being effective. The fermentation liquor, freed of mycelia can be extracted with water-immiscible solvents. The extracts can be combined, either before or after washing with an alkaline solution, illustratively sodium bicar- 6. bonate, suitably dried, as for example, over anhydrous sodium sulfate, and the resulting purified fermented steroid obtained by recrystallization from organic solvents, by trituration with organic solvents or by chromatography in order to isolate the thus obtained steroids from the other fermentation products.

The following examples are illustrative of the process of the present invention and are not to be construed as limiting.

EXAMPLE 1 Progesterone Twelve liters of medium consisting of one percent Cerelose dextrose, two percent cornsteep liquor of sixty percent solids, was adjusted to pH 4.9 with sodium hydroxide. Ten milliliters of lard oil containing 0.1 to 2.0 percent octadecanol was added to prevent foaming. The medium was steam sterilized at fifteen pounds pressure for thirty minutes. Upon cooling, the sterile medium was inoculated with a 24hour growth, from spores, of Septomyxa a ffinis ATCC 6737. The medium was agitated, and sparged with sterile air at the rate of one liter of air per minute. After culturing at room temperature for 24 hours, the pH was 7 .35. To this 24-hour culture there was added six grams of progesterone dissolved in 130 milliliters of acetone. Fermentation of the progesterone was maintained for 24 hours, at which time the pH was 8.1. The fermentation broth was strained through gauze to separate the mycelium. The mycelium was washed once with one liter of acetone and then twice with oneliter portions of methylene chloride. The acetone and methylene chloride washings were combined with an additional four liters of methylene chloride and this seven liters of extract and solvent was then used to extract the filtered beer. Upon separation of the extract from the beer, the beer was twice more extracted Withthree-liter volumes of methylene chloride. All of the acetone and methylene chloride extracts were combined and washed with 1200 milliliters of two percent sodium bicarbonate solution, and then with 1200 milliliters of water. The washed solvent extract was then dried with anhydrous sodium sulfate and vacuum evaporated to leave seven grams of extract.

The extract was dissolved in 230 milliliters of benzene and fractionated over a column of 300 grams of alumina (acid washed, dried at degrees centigrade) using 230-milliliter portions of developing solvent as shown in Table I.

benzene .610."

benzenepercent ether o benzene10 percent ethe 27- chloroform-20 percent acetone.

28... chloroform-50 percent acetone 771 29 acetone 52 30 methanol 35 to give 258 ,,gram .of re idu w i h wastr tuta d 1'. h

'7 filtra e s l ds freedoms 8 t reugh'fl3 wer c mbined eth r, and ee h r e an ed, un t e de an e p s colorl ss- Th s l t 4 5 gram .Q Li -androstadi ener- ,-l7.-.d one. having a melting p in of 143 to 144.5 degrees centigrade, [a] of plus 115 degrees AQiZ- 239.5 mg (E=-12,500-) This compound is known to be useful in the production of estrone.

Eluate solids fractions -19 through 25 were combined and reerystallized twice from one milliliter of ethyl acean 2 Yi d 9-3948 a of fi-1 Y Q Y A- B IQ l slis e-nnet de dmt sw mne) hav g a meltin a t st r '1 to 7. de ee en israd [easto plu 2 gig s ers 7 X3; 243 m .(E 16,000)

slurried with 100 grams of Celite diatomaceousearth and then the non-aqueous phase was added and the mixed slurry was poured into a column. The surplus non-aquepus phase was drained from the column. This column,

topped with the vdiatomaceous earth-steroid residue was then eluted with thirteen ZOO-milliliter portions of the non-aqueous phase obtained above, 200 milliliters of methanol, and 200 milliliters of methylene chloride.

fractions 4 through 11 were l-dehydrotestosterone' Fractions 14 and 15 were l-dehydrotestololactone. The combined residue of fractions 14 and 15 was taken up in methanol, decolorized with activated carbon, evaporated and recrystallized from two milliliters of ethyl acetate to yield pure l-dehydrotestololaetone having a melting point {i 221 t 22- de r s sn i d nd sha ts is i i f red spectra and optical rotation.

AM LE 2 Progesterone The fermentation and extraction procedure of Example 1 followed, except that at the time of the progesterone addition, forty milliliters of H PO .was added to a i it th ee f m PH -3 to p The total resulting extract of six grams was dissolved in benzene and ahmniatc ra he eq e a i E ample 1. j Eluate solids fractions 8 through 16 (3.34 grams) were 'combined and triturated with ether to yield 1.94 grams of re amely Aand st d snc-iUn ons hav n a rr elting point of 143.5 to 144.5 degrees centigrade.

EXAMPLE 3 1 7o,-2] -dihydr0xy-4-pregnene-3,20 di0ne The fermentation and extraction procedure of Example was followed, except that the steroid substrate progesterone was replaced by two grams of '17a,2-1-dihydroxy- ,4-pregnene.-3,20-.dione dissolved in 1.00 milliliterslof ,rnethanol.

The extractives weighed three grams. The extractives were dissolved in 3.6.0 milliliters of ethylene dichloride and fractionated over a column of 240 grams of Florisil :il sil fia s w th. 60-. n 1l l t r p r n of solvent as follows: ethylene dichloride, three portions; ethylene 'was permitted to separate. The aqueous phase was then removed. A 15.0-milliliter portion of the'aqueous phase dish o ir easeta e 2551. tw pardo s; 5 1 th ee 111% iansfl la ihJfi-PQE. as; 1192 h e meter ra., hs 2 t9 4); 3;; hree P9 i Ql1 v(t act sz i 1 9 1. .521 pen se three en se assi ns p tions 1 51 mth asl. e versi s- Elva e s9 ist 2 m 7 wins a me tin r 9f ees Otherwise like Example 1, allopregnane-3,lLZQtflene was used in p a e o o st rone. to produ e e-an sne-3l r -t io e ha n adrenoe tic 1 mm. IiYitY nd eni and f t o e i act vi y A small amount contaminating pregnane-ELILZO tri ne iri the Sta t ma e ial es te n fi' i m. 1 Q1 .1 .17 one hav s me trqn sact ity- EXAMPLE 6 1 -desox ycprticosterone Fo owin Ex mple usin 1 esoxwr is stsraae n Plea 9 n oseste qne Pr du d l-de rq-ltb qor sten n -deh drotq tq teran A-ansl sta .3 1 :$1 9l1 and rdehyt ro es alqh ne- EXAMPLE 7 sindros tenediorre Following Example 1, using .4-androstene-3, 1'7.=dione, in place of progesterone, produced l-dehydrotestosterone, 1,4 androstadiene 3,17 dione, and 1 dehydrotestoloh actone.

' EXAMPLE 8 .T tq mne :E lIQW II Example u in .tcst te e e plays progesterone, produced mainly l-dehyglrotestostergne, and a so flzan rqstadiene-fi 1 mm- ExA PLn 9 P qgq em Sepzemyxa afiinis was .grown for eighteen hours in nu trient medium prepared as described in Example 1. i The mycel-ium'was centrifuged from the medium and washed six times by centrifugation with distilled water. The washed rnyceliuin was suspended in .600 milliliters of dis: tilled water and divided into six portions in 25.0rnilliliter Erlenmeyer flasks. Additions were made as indicated in Table II, and forty milligrams of progesterone dissolved in one milliliter of acetone was added to each flask. The flasks were shake cultured at room temperature for 24 hours. 'Each whole beer was then extracted four times with 25-mi-ll-iliter portions of methylene chloride. Each extract was then washed with two percent sodium bicarbonate solution and distilled water, dried over anhydrous sodium sulfate, and the solvent then evaporated. The residues were analyzed by paper chromatography with results as shown in Table II. The 'l-dehydropro'gesterone exhibits enhanced progestational activity.

In the same manner replacing .the progesterone sub: strate by -1=9-normethyltestosterone in conjunction with IhO OL g Y Wa hed cells buffered at pH 7 produced 1,- Qshrdm-Hm m th ltes ostero e which an be er.-, a e in asisl to es asliol.

TABLE II 1311 Gamma isolate per 200 gamma extract Additives l-dehydrol-dehydrol-dehydrol-dehydro- Initial Final progester- 4-androtestostestololacone stene-3,17- terone tone dione 3. 9 6. 9 60 2O 12 7. 6 8.0 KHaCOa 60 6 10 2.0 2. 1 HaPO4 75 2 6 3. 9 3. 9 dextrose cerelose. U 50 40 2 6. 2 6. 4 09003 glu- 8 50 40 2 cose. 5. 8 7. phosphate 2 60 30 1b buffer.

EXAMPLE 10 tals melting at 158 to 161 degrees centigrade. Recrystal- 1 118,21 dihydroxy-4,1 7 -pregnadiene-3-0ne Septomyxa afiinis was maintained on malt agar slants composed of fifty grams of dry malt extract, five grams of Edamine enzymatic digest of lactalbumin, and twenty grams of agar diluted to one liter with tap water and ad justed to a pH between 6.5 to 7.0. Inoculum from the agar slant was transferred to one-liter Erlenmeyer flasks containing 100 milliliters of malt extract agar and incubated at room temperature for from four to seven days to produce spores. The spores were suspended in 100 milliliters of sterile one percent saline. Five milliliters of this saline spore inocnlum was introduced into each of five 250-milliliter flasks containing 100 milliliters each of medium, as in Example 1, which was adjusted to pH 4.8 to 5.0. These were incubated for 48 to 72 hours with shaking at room temperature and then added to six liters of medium as in Example 1. This culture was maintained for 24 hours at room temperature with aeration at a rate of one liter per minute. This six liters of seed culture was then added to 100 liters of medium as in Example 1 and grown at room temperature for 24 hours with agitation and aeration at a rate of two liters per minute. To this 24-hour culture there was added 25 grams of 11,8,2 l-dihydroxy-4,l7(20) -pregnadiene-3-one dissolved in 500 milliliters of acetone. Agitation and aeration at the rate of one liter per minute was continued for 24 hours.

The broth was filtered to give mycelium and filtered beer. The mycelium was washed with twenty liters of water and the water washing was added to the filtered beer. The washed mycelium was suspended and slurried twice each time with twelve liters of acetone, and then suspended and slurried twice each time with twelve liters of methylene chloride. The thus obtained acetone and methylene chloride mycelium extracts were pooled and added to the extract obtained from four extractions of the filtered beer and water wash, each extraction being with 24 liters of methylene chloride. The extracts from the filtered beer and water wash and those from the mycelium were combined and washed twice each time with twelve liters of two percent sodium bicarbonate solution and then twice each time with twelve liters of water. The washed extract was concentrated in a still to give three liters of concentrate which was then evaporated to dryness on a steam bath in air to give 54.3 grams of crude crystalline residue. This residue was triturated six times, each time with 25 milliliters of diethyl ether. The remaining crystals weighing 20.9 grams were dissolved in 250 milliliters of hot methanol, filtered, and cooled to room temperature to give 11.7 grams of crystals melting at 181.5 to 183.5 degrees centigrade. A 305-milligram portion of these crystals was dissolved in six milliliters of boiling ethylene dichloride. The hot solution was filtered and crystallized at room temperature followed by chilling at about five degrees centigrade to complete crystallization. There was thus obtained 238 milligrams of cryslization, by the same technique, produced 215 milligrams of 1113,21 dihydroxy 1,4,l7(20) pregnatriene 3 one having a melting point of 149 to 153 degrees centigrade. Acetylation of the 2l-hydroxy group with acetic anhydride in pyridine is productive of llfl-hydroxy-21-acetoxy- 1,4,17(20)-pregnatriene-3-one which is converted, with two molar equivalents of hydrogen peroxide in dry tertiary butyl alcohol preferably containing about a molar equivalent of pyridine and in the presence of a catalytic amount of osmium tetroxide, to 11 3,17ot-dihydroxy-2l-acetoxy- 1,4-pregnadiene-3,20-dione.

The 11.7 grams of crystals from methanol, having a melting point of 181.5 to 183.5, were 1 methoxy 115,21- dihydroxy 4,17(20) pregnadiene 3 one produced from 11fl,21 dihydroxy 1,4,17(20) pregnatriene 3- one by the action of hot methanol. Using less rigorous conditions or other solvents such as ethyl acetate is productive of 1113,21 dihydroxy 1,4,l7(20) pregnatriene- 3 one, the 1 methoxy compound not being formed.

EXAMPLE 11 17cc,21 dihydroxy 4 pregnene 3,11,20 trione Otherwise in accordance with both Examples 3 and 10, using 170:,21 dihydroxy 4 pregnene 3,11,20 trione as steroid substrate produced 170;,21 dihydroxy 1,4- pregnadiene 3,11,20 trione, having anti-inflammatory and antiarthritic properties. Either the free product or 21-esters such as for example the 21-acetate or propionate is thus useful therapeutically.

EXAMPLE 12 1 I 18,] 711,21 trihydroxy 4 pregnene 3,20 dione Otherwise in accordance with both Examples 3 and 10, replacing the starting steroid substrate by 1.1 ;9,17a,21 trihydroxy 4 pregnene 3,20 dione produced 1l/3,l7a,21 trihydroxy 1,4 pregnadiene 3,20 dione which in its free form or as the 2l-esters such as the ZI-acetate or 21- propionate demonstrated anti-inflammatory and antiarthritic properties qualitatively similar to those of hydrocortisone.

EXAMPLE 13 11 ketoprogesterone Otherwise like Example 1, substituting 11 ketoprogesterone as the starting steroid substrate produced the anabolically active compounds l-dehydroadrenosterone, 1 dehydro ll ketotestosterone and l dehydro 11- ketotestosterone and 1 dehydro l1 ketoprogesterone, having glucocorticoid activity.

EXAMPLE 14 11a hydroxyprogesterone Otherwise like Example 1, substituting 11a hydroxyprogesterone as the starting substrate produced the ana bolically active compounds 1 dehydro 11oz hydroxytestosterone, 11a hydroxy 1,4 androstadiene 3,17- dione and 1 dehydro -hydroxyprogesterone.

EXAMPLE 15 11/8 hydroxyprogesterone Otherwise like Example 1, substituting 115 hydrox-yprogesterone as the starting substrate produced the anabolically active compounds 1 dehydro 115 hydroqr-ytestosterone, 11p hydroxy 1,4 androstadiene' 3,17- dione and 1 dehydro 11 3 hydroxyprogesterone, having glucocorticoid activity.

EXAMPLE 16 19 normethyltestosterone Otherwise like Example 1, substituting 19 norme thyltestosterone as the starting steroid produced estrone and estradiol.

EXAMPLE 17 .Qthenwise like Example 1, substituting -1-7oe methyltestosterone as the starting steroid produced 1 dehydro 17,0: methyl.testosterne .of enhanced anabolic activity.

Following the procedures of Examples 1 through 17 using Septomyxa "qescu'li, Se-ptomyqca "corni, Sept omyxa salicina or Septomyrgz tyl asl ze i produced the results as obtained with Septomyxd affinis. i i

hemp? 9 6.0L methylhydxocontisone fitherwise li e Exam l 1, Q rhethy si qho hsoh w fli h P h 0 Pr9e9st 9 9 t rhsihee 1 deh dro- 6d methylhydrocortisoige.

' i h' indqr t hd hat the inv n i n i n t to h limited to the and d ai s 9.? q hrh ieh r .es q 9 pounds show-n and described, as obvious modifications and equivalents will be apparent to those skilled in the art, and e ihx nt hh i t h hfh h to e l PiF -9I Y b the shap of the appended clair ns.

" 'We claim:

1. A process for the ll-dehydrogenation of a steroid which comprises contacting a viable fungus of the genus Septom-yxawith a 3-oxygenated steroid in which the l,-2,-3-position carbon atom group is selected from z z- H2 2*- JHQH 23n --CH --CH(CH )CO under aerobic conditions.

2. A process for the I Z-dehygimgenatiQn of a steroid hi h h h is shhtahtin a viah e .fhhshs M the en SeptomyXawith a 1,2-trih dro3-0Xygenated steroid under aerobic fermentation conditions.

3. A .process for the l,2.-.dehydrogenation of a steroid which comprises contacting a viable fungus of the genus Septomyxa with a l,2-tetrahydro-3oxygenated steroid under aerobic fermentation conditions.

.4. A process for the l,-2-dehydrogenation of a steroid which comprises contacting Septomyxa afiinis with a 3- oxygenated steroid in which the 1,2,3-position carbon atom group is selected from' -CH CH CO, CHZ Z HOH and mQHF 3 under aerobic conditions.

5. A process for the preparation of -l-dehydro steroids which comprises contacting a 4-dehydro-3-ke to steroid selected frorn'the pregnane series and the andro sta ne series compounds containing a l,'2-t rihy dro linkage under aerobic fermentation conditions with a fungus of the genus Septomyxa in the presence of a nutrient medium.

6. A process for the ;1,g-dehydrogenation of a 20-oxygenated steroid which comprises growing a fungus of the genus Septornyxa under aerobic conditions in the presence of a nutrient medium and a ZO-oXygenated steroid satumihd at the -1: and 2carbon atoms until substantial 1,2.- dehydrpgenation has occurred.

- 7. A process for the 1,2-dehydrogenation of a 20-oxygenated steroid which comprises growing a fungus ofthe "12 genus Septornyxa under aerobic conditions in the presence of a nutrient medium containing assimilable non-steroidal en er an gt gog gnna steroid saturatedfafflie "'1?- and 2-carbon atoms until substantial 1,2-dehydrogenation nhgsgclcfiirjrdg L i 8. A process for the 1,2-dehydrogenatig n of a ggrigeto steroid which comprises growing a fungus of the genus .SEPFQIPYPE? hash; EJQIPPi? hhhh iiehs in h M9399??? 9 a nutrient medium containing assimilable non-steroidal car.- h n a 20-k to st r d satur ted a t e 1- a Z- r: zbon atoms until substantial l g-dehydrogenation has oc; curred. i

9. A process for the 1,2-dehydrogenation of a steroid comprising growing a fungus 'of the genus Septomyxa .under submerged aerobic agitated fermentation .condi= tions in the presence of a 1,2-tetrahydro-3-oxygenated steroid and separating the resulting 1-dehydro-3-oxygenated steroid.

10. A process for the 1 ,Q-dehydrogenation of a steroid hih hih s owin seatom rh .rlfi i hhs h shhhihxs ae b a ta ed fermen ati n S E i Q9 h a h eh; medium P IWQ E E a 1 1 ah sh9- hx shnate smile and separat ng the hhhhlhhg -s1eh ih =9 eased steroid.

11- Th PFQFFSS h .Qlaim wherein th h h-. hh hi lihh shh h hs i hi h lh hqh-s sihia ha hph r 13- A hhss thith :hx -si h slm hhht hh ri e e nih s m ris hg g w g a tf h h 9 th 8 25. .S PthihYith unde subm rged hewh h ash h shhhit hh i a he.- dihm coht hihs a ,LZ-thh x h -h shhxy teroid and ss h atihs the resu ng -dhh d hfi-hxy enated tamin- 14- ,A rh ss sth hhme gr ingathh h .Q thesh ns e thmy h unde aerhhi PQ t Qh in a mq ihm honta h he p o hste qh a d he .ih t e r su tin 1.2m.- hydro" steroid. i

5- A PI9 sh h i h g h t he haw-rm Jfit i hh h lub hrhed ae hhih a t ted hhh qn in a medium hh i h h s ha h hyd a d h hs h hhh ahd'separ l n th resul ng LZrdPhY FQ s er d- 16. A process comprising growing a fungus of the genus -P UJYX? nder a h hi hhhitihhs it; a .13. !1 3 -,h h. ng .-%2; =h hn r h-amsnerreiflhi aha h hha hs esu in -tdehyd h thihi 7 A" .ss h m r s ns' ston n d pt m fi nis under su e d hh hh h a itated .h h a me.- dihm .crin a ihs h hh yshat @2 5 .1 'h-nhw .-n e- 3,2O:dit hi= I hd eeh tih the resultin 1 dehydro steroid. A

.A p ams h mh i ih vsrhw hs a i l l h 0. the us S qh x u d r aq i he mit-i n i a medium .wn a n e 1mlhxdhqh .ll (20l r eshahihnweqne a s a at n he h i tih .lllml -di nhpx- 1 4,11- 0 -1 na in i3--Qhh;

.-Am9hh$ hhmh hih awn n hi wmm Mini; de ubme e ahrhl a i hie .6011 qh .i-h hhue um 9 wining hh h drat ahs 1 312. h r etr17 2h s 13 genus Septomyxa under aerobic conditions in a medium containing an allopregnane-ZS-one and separating the resulting 1,2-dehydro steroid.

24. A process comprising growing a fungus of the genus Septomyxa under aerobic conditions in a medium containing 11a-hydroxyallopregnane-3,ZO-dione and separating the resulting 1,2-dehydro steroid.

25. A process comprising growing Septomyxa affinis under submerged aerobic agitated conditions in a medium containing carbohydrate and 11u-hydroxya1lopregnane-3,20-dione and separating the resulting 1,2-dehydro steroid.

26. A process for the production of 1-dehydro-6-methylhydrocortisone which comprises: growing a microorganism of the genus Septomyxa in a nutrient medium containing assimilable non-steroidal carbon, nitrogen and phosphorus, and 6-methylhydrocortisone, and recovering the thus-produced 1-dehydro-6-methylhydrocortisone.

References Cited in the file of this patent Thorn: Ann. New York Academy of Science, 60, 1 October 1954, pages 5, 24 and 26.

Ainsworth et a1.: Iour. Gen. Microbiology, vol. 10,

15 1954, pages 465-474. 

1. A PROCESS FOR THE 1,2-DEHYDROGENATION OF A STEROID WHICH COMPRISES CONTACTING A VIABLE FUNGUS OF THE GENUS SEPTOMYXA WITH A 3-OXYGENATED STEROID IN WHICH THE 1,2,3-POSITION CARBON ATOM GROUP IS SELECTED FROM -CH2-CH2-CO-, -CH2-CH2-CHOH-, AND -CH2-CH(CH3)-CO- UNDER AEROBIC CONDITIONS. 